ES2339953T5 - O-linked factor VII glycoforms and method of manufacture - Google Patents

Abstract

The present invention relates to compositions comprising glycoproteins having altered patterns of O-linked glycosylation, in particular Factor VII, Factor IX, and methods for making these.

Description

DESCRIPTION

O-linked factor VII glycoforms and method of manufacture

FIELD OF THE INVENTION

[0001] The present invention relates to compositions comprising factor VII polypeptides that have altered patterns of O-linked glycosylation.

BACKGROUND OF THE INVENTION

[0002] The biological activity of many glycoproteins is highly dependent on the presence or absence of particular oligosaccharide structures attached to the glycoprotein. The glycosylation pattern of a therapeutic glycoprotein can affect numerous aspects of therapeutic efficacy, such as, e.g. eg, solubility, resistance to proteolytic attack, thermal inactivation, immunogenicity, half-life, bioactivity, bioavailability, and stability.

[0003] Glycosylation is a complex post-transitional modification that is cell dependent. After translation, the proteins are transported to the endoplasmic reticulum (ER), glycosylated, and sent to the Golgi for further processing and subsequent targeting and / or secretion. During glycosylation, either N-linked or O-linked glycoproteins are formed.

[0004] Serum proteins involved in coagulation or fibrinolysis, including, eg. eg, factor VII and factor IX are proving useful for therapeutic agents to treat a variety of pathological conditions. Accordingly, there is an increasing need for formulations comprising these proteins that are pharmaceutically acceptable and show uniform and predetermined clinical efficacy.

[0005] Due to the many disadvantages of using human plasma as a source of pharmaceuticals, it is preferred to produce these proteins in recombinant systems. Coagulant proteins, however, are subject to a variety of co- and post-translational modifications, including, eg. eg, asparagine-linked glycosylation (N-linked); glycosylation linked to serine or threonine (linked to O); and the carboxylation of glu residues. These modifications can be qualitatively or quantitatively different when heterologous cells are used as hosts for large-scale protein production. In particular, production in heterologous cells frequently results in a different set of glycoforms, the identical polypeptides of which have different covalently linked oligosaccharide structures.

[0006] In different systems, variations in the oligosaccharide structure of therapeutic proteins have been linked to, inter alia, changes in immunogenicity and space in vivo. Thus, there is a need in the art for compositions and methods that provide preparations comprising recombinant human factor VII or modified factor VII or factor VII related polypeptides containing predetermined glycoform patterns.

SUMMARY OF THE INVENTION

[0007] The present invention relates to a pharmaceutical composition comprising (a) a preparation comprising human factor VII polypeptides exhibiting predetermined serine-linked glycoform patterns and b) a pharmaceutically acceptable carrier.

[0008] As used in this case, a glycoform model refers to the distribution within the preparation of oligosaccharide chains having variable structures that are covalently linked to a serine residue located in an EGF-like domain in the skeleton amino acid content of the polypeptide.

[0009] The human factor VII polypeptide containing a Cys-X1-SerX2-Pro-Cys motif and where said serine is located at position Ser52 and is part of a Glc-O-Ser covalent bond, where at least 85 % of said serine 52 residues in the preparation are glycosylated with Xyl-Xyl-Glc.

[0010] In an embodiment of the invention, at least 90%, at least 95% or at least 98% of said serine residues are glycosylated with Xyl-Xyl-Glc.

[0011] In another aspect, the invention provides methods for the manufacture of factor VII preparations as described below. The methods are useful for remodeling or altering the glycosylation pattern present in a factor VII polypeptide in its initial expression.

[0012] As described below, a general enzymatic methodology for the modification of glycans (particularly O-linked glycans) of factor VII polypeptides, to improve or enhance their properties pharmaceuticals. One method involves treating factor VII polypeptide with xylosidases to remove any terminal xylose residue; other methods include fixing xylose residues to exposed glucose or xylose residues in the factor VII polypeptide by treatment with xylosyltransferases; a third method includes the binding of glucose residues to serine and / or threonine amino acid residues in factor VII, the backbone of the polypeptide thus creating a glycosylated factor VII polypeptide. The invention in particular provides a method as set forth in claim 3.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]

Fig. 1 shows the glycosylation of serine 52 of factor VII-wt.

Fig. 2 shows a mapping of factor VII O-glycosylation.

Fig. 3 shows a reaction scheme for the manufacture of a glycoprotein preparation that exhibits a predetermined serine / threonine linked glycosylation.

Fig. 4 shows a chromatogram of first fractions "A" and "B" showing the HIC cycle.

Fig. 5 shows a chromatogram obtained by reloading fraction "A" on the HIC column; Glc-O-Ser52-FVII was identified in the maximum value fraction, fraction 10.

Fig. 6 shows a chromatogram obtained by reloading fraction "B" on the HIC column; Xyl-Xyl-Glc-O-Ser52-FVII was identified in the maximum value fraction, fraction 15.

Fig. 7A shows a tryptic peptide map of the maximum value fraction, fraction 10; the arrow indicates the Glc-O-Ser52 O glycopeptide. Fig. 7B shows a tryptic peptide map of the maximum value fraction, fraction 15; the arrow indicates the glycopeptide Xyl-Xyl-Glc-O-Ser52 O.

Fig. 8A shows an analysis of the total mass of the maximum value fraction, fraction 10; the arrow indicates the Glc-O-Ser52-rFVIIa O glycoform.

Fig. 8B shows an analysis of the total mass of the maximum value fraction, fraction 15; the arrow indicates the Xyl-Xyl-Glc-O-rFVIIa O glycoform.

DETAILED DESCRIPTION

[0014] The following abbreviations are used here:

Glc = glucosyl

Xil = xilosilo

Ser = serina (one letter code: S)

Thr = threonine (one letter code: T)

Pro = proline (one letter code: P)

Cys = cysteine (one letter code: C)

FVII = factor VII

FVIIa = activated factor VII (double-stranded)

FIX = factor IX

FIXa = activated factor IX (double-stranded)

[0015] As used herein, a "glycoform model" (or "glycosylation model") refers to the distribution within the preparation of oligosaccharide chains having variable structures that are covalently linked to a serine residue Located in the amino addition backbone of the polypeptide.

[0016] "Homogeneity" refers to structural consistency across the population of conjugated glycan polypeptides. Thus, a glycoprotein preparation is said to be approximately 100% homologous if all of the glycoprotein molecules contained contain identical glycans attached to the relevant glycosylation site. For example, a preparation of factor VII polypeptides is said to be at least 90% homologous if at least 90% of the factor VII polypeptide molecules contain the glycan of interest bound to serine 52 (eg. , Xyl-Xyl-Glc-O-Ser52).

[0017] "Uniform glycoform" or "uniform glycosylation" or "uniform glycosylation pattern", when referring to a species of glycopeptide, refers to the percentage of acceptor fractions, that is, serine residues, which are glycosylated by glycan of interest. For example, in the case of factor VII, substantially uniform glycosylation patterns exist if substantially all (as defined below) serine residues at position 52 are glycosylated with the glycan of interest. It is understood by those skilled in the art that the raw material may contain glycosylated serine residues that are glycosylated with species having the same structure as the glycan of interest. Thus, the calculated percentage of glycosylation includes the serine residues that are glycosylated with the glycan of interest according to the invention, as well as those glycosylated serine residues already with the glycan of interest in the raw material.

[0018] At least about 85%, such as at least about 95%, or at least about 98% of the serine residues in the factor VII polypeptide are glycosylated with a predetermined and specific glycan or a glycan of interest . The glycosylation pattern is usually determined by one or more methods known to those skilled in the art, such as, e.g. eg, tryptic digestion (HPLC) followed by high performance liquid phase chromatography, liquid chromatography-mass spectrometry (LC-MS), flight spectrometry matrix assisted laser desorption mass time (MALDITOF), capillary electrophoresis, and the like.

[0019] The term "acceptor fraction" is intended to encompass the group or fraction to which a desired group of oligo- or monosaccharides is transferred such as, without limitation, the serine residue located within a Cys-X1-Ser model -X2-Pro-Cys, a Glc residue covalently linked to such a serine residue or an Xyl residue covalently linked to a Glc residue or a Xyl residue in a Glc-O-Ser or Xyl-Glc fraction -O-Ser, respectively.

[0020] The term "saccharide donor fraction" is intended to encompass an activated saccharide donor molecule (eg, a desired oligo- or monosaccharide structure such as, for example, a xylosiloxylosyl donor, a xylosyl donor , or a glycosyl donor) that has an exit group (eg, UDP-xylose or UDP-glucose) suitable for the donor fraction that acts as a substrate for the relevant catalytic enzyme (eg, glycosyltransferase, xylosidase or xylosyltransferase).

[0021] Oligosaccharides are considered to have a reducing end and a non-reducing end, even the saccharide at the reducing end is in fact a reducing sugar. In accordance with accepted nomenclature, oligosaccharides are represented here with the non-reducing end on the left and the reducing end on the right (eg Xyl-Xyl-Glc-O-Ser).

[0022] As used herein, the term "glycan" or, interchangeably, "sugar chain", "oligosaccharide chain" or "oligosaccharide moiety" refers to the entire structure of oligosaccharides that is covalently linked to a sole serine residue. The glycan can comprise one or more saccharide units; Examples of glycans include, e.g. Eg, Glc-, Xyl-Glc-, and Xyl-Xyl-Glc-.

[0023] The term "O-glycosylation site" is intended to indicate the serine (ie, Ser) glycosylation site located within the Cys1-X1-Ser-X2-Pro-Cys2 motif where Cys1 and Cys2 are the first and the second conserved cysteines of the EGF repeat and X1 and X2 is independently any amino acid. These include the glycosylation site at the Ser-52 (S52) position of human wt-FVII and the corresponding residues in homologous polypeptides such as, without limitation, FVII sequence variants and FIX polypeptides. The term "corresponding residues" is intended to indicate the amino acid residue Ser corresponding to the Ser52 residue of wild-type factor VII (see Fig. 1) when the sequences are aligned. Amino acid sequence homology / identity is conveniently determined from aligned sequences, using a suitable computer program for sequence alignment, such as, e.g. eg, the ClustalW program, version 1.8, 1999 (Thompson et al., 1994, Nucleic Acid Research, 22: 4673-4680). For example, the Ser52 residue of factor VII-wt corresponds to the Ser53 residue of factor IX-wt. Furthermore, it should be understood that polypeptide variants can be created containing non-naturally occurring Cys-X1-Ser-X2-Pro-Cys motifs and thus containing non-naturally occurring O-glycosylation sites that can be glycosylated according to the present invention. In one embodiment of the invention, the O-glycosylation site is a serine glycosylation site and the motif is Cys1-X1-Ser-X2-Pro-Cys2.

[0024] The term "terminal glucose" is intended to comprise linked glucose residues such as the terminal sugar residue in a glycan, or the oligosaccharide chain, ie, the terminal sugar of each antenna is glucose. The term "terminal xylose" is intended to encompass linked xylose residues such as the terminal sugar residue in a glycan, or oligosaccharide chain.

Enzymes

[0025] Protein O-glycosyltransferase can be prepared as described, e.g. eg, in Shao et al. (Glycobiology 12 (11): 763-770 (2002)).

[0026] Alpha-xylosidase enzymes can be prepared, eg. eg, as described by Monroe et al. (Plant Physiology and Biochemistry 41: 877-885 (2003)).

[0027] The enzyme, UDP-D-xylose: p-D-glucoside α-1,3-D-xylosyltransferase can be prepared from HepG2 cells as described by Omichi et al. (Eur. J. Biochem. 245: 143-146 (1997)).

[0028] The enzyme, UDPD-xylose: α-D-xyloside α-1,3-xylosyltransferase can be obtained from HepG2 cells as described by Minamida et al. ((J. Biochem. (Tokyo) 120: 1002-1006 (1996)).

[0029] UDP-beta-D-glucose is commercially available from, eg. eg Sigma (Sigma U4625)

[0030] UDP-D-xylose is commercially available from, eg. eg Sigma (Sigma U5875)

Glycoproteins

[0031] The motif: Cys-X1-Ser / Thr-X2-Pro-Cys appears to be mainly found in multimodular protein domains of epidermal growth factor (EGF) such as coagulation factors and fibrinolytic factors. The reason is a consensus sequence for O-glucose modification whereby a serine glucose (Gic-O-Ser) or threonine glucose (Glc-O-Thr) bond is formed. Clotting factors VII, IX, X and XII as well as plasma protein Z, fibrillin and thrombospondin have all been shown to contain the consensus sequence Cys-X1-Ser / Thr-X2-Pro-Cys. Of these, factors VII and IX and protein Z have been described as containing the Cys-X1-Ser-X2-Pro-Cys consensus sequence.

[0032] Factor VII (proconvertin) is a vitamin K-dependent serine protease that participates in the blood clotting process to participate in the activation of prothrombin in thrombin. FVII is activated in FVIIa by contact with exposed tissue factor (TF) at wound sites on the vessel wall. Factor VII polypeptides and factor VII related polypeptides

[0033] As used herein, the terms "factor VII polypeptide" or "FVII polypeptide" means any protein comprising the amino acid sequence 1-406 of wild-type human factor VIIa (ie, a polypeptide having amino acid sequence disclosed in US Patent No. 4,784,950), variants therein as well as Factor VII related polypeptides, factor VII derivatives and factor VII conjugates.This includes FVII variants, related polypeptides with Factor VII, factor VII derivatives and factor VII conjugates that exhibit the same or improved biological activity relative to wild-type human factor VIIa.

[0034] The term "factor VII" is intended to comprise factor VII polypeptides in their undivided (zymogen) form, like these that have been proteolytically processed to produce their respective bioactive forms, which may be designated factor VIIa . Typically factor VII is cleaved between residues 152 and 153 to produce factor VIIa. Variants of factor VII of this type can exhibit different properties relative to human factor VII, including stability, phospholipid binding, altered specific activity, and the like.

[0035] As used herein, "wild-type human FVIIa" is a polypeptide having the added amino sequence disclosed in US Patent No. 4,784,950.

[0036] The term "derived factor VII" as used herein is intended to designate an FVII polypeptide that exhibits the same or improved biological activity relative to wild-type factor VII, where one or more of the additions of amines the parental peptide has (have) been genetically and / or chemically and / or enzymatically modified (s), e.g. ex. by alkylation, glycosylation, PEGylation, acylation, ester formation or amide formation or the like. This includes but is not limited to PEGylated human factor VIIa, PEGylated human cysteine factor VIIa and its variants. Non-limiting examples of factor VII derivatives include derivatives of GlyPEGylated FVII as disclosed in WO 03/31464 and US patent applications US 20040043446, US 20040063911, US 20040142856, US 20040137557, and US 20040132640 (Neose Technologies, Inc.) ; FVII conjugates as disclosed in WO 01/04287, US patent application 20030165996, WO 01/58935, WO 03/93465 (Maxygen ApS) and WO 02/02764, US patent application 20030211094 (University of Minnesota).

[0037] The term "enhanced biological activity" refers to FVII polypeptides with i) the same proteolytic or increased activity compared to recombinant wild-type human factor VIIa or ii) to FVII polypeptides with substantially the same binding activity to TF or increased compared to recombinant wild-type human factor VIIa or iii) to FVII polypeptides with substantially the same half-life in blood plasma or increased compared to recombinant wild-type human factor VIIa. The term "PEGylated human factor VIIa" means human factor VIIa, with a PEG molecule conjugated to a human factor VIIa polypeptide. It should be understood that the PEG molecule can be attached to any part of the factor VIIa polypeptide including any amino acid residue or carbohydrate moiety of the factor VIIa polypeptide. The term "PEGylated cysteine human factor VIIa" means factor VIIa with a PEG molecule conjugated to a sulfhydryl group of a cysteine introduced into human factor VIIa.

[0038] Non-limiting examples of factor VII variants that have the same or increased proteolytic activity compared to recombinant wild-type human factor VIIa include S52A-FVIIa, S60A-FVIIa (Lino et al., Arch. Biochem. Biophys. 352: 182-192, 1998); Variants of FVIIa exhibiting increased proteolytic stability are disclosed in US Patent No. 5,580,560; factor VIIa that has been proteolytically cleaved between residues 290 and 291 or between residues 315 and 316 (Mollerup et al., Biotechnol. Bioeng. 48: 501-505, 1995); oxidized forms of factor VIIa (Kornfelt et al., Arch. Biochem. Biophys. 363: 43-54, 1999); FVII variants as disclosed in PCT / DK02 / 00189 (corresponding to WO 02/077218); and FVII variants that exhibit increased proteolytic stability as disclosed in WO 02/38162 (Scripps Research Institute); FVII variants that have a modified Gla domain and that exhibit improved membrane binding as disclosed in WO 99/20767, US Patents US 6017882 and US 6747003, US Patent Application 20030100506 (University of Minnesota) and WO 00/66753, US Patent Applications US 20010018414, US 2004220106, and US 200131005, US Patents US 6762286 and US 6693075 (University of Minnesota); and FVII variants as disclosed in WO 01/58935, US Patent US 6806063, US Patent Application 20030096338 (Maxygen ApS), WO 03/93465 (Maxygen ApS), WO 04/029091 (Maxygen ApS), WO 041083361 (Maxygen ApS), and WO 04/111242 (Maxygen ApS), as in WO 04/108763 (Canadian Blood Services).

[0039] Non-limiting examples of FVII variants that have increased biological activity compared to wild-type FVIIa include FVII variants as disclosed in WO 01/83725, WO 02/22776, WO 02/077218, PCT / DK02 / 00635 (corresponding to WO 03/027147), Danish patent application PA 200201423 (corresponding to WO 04/029090), Danish patent application pA 2001 01627 (corresponding to WO 03/027147); WO 02/38162 (Scripps Research Instiute); and FVIIa variants with enhanced activity as disclosed in JP 2001061479 (Chemo-Sero-Therapeutic Res Inst). Examples of factor VII variants include, without limitation, L305V-FVII, L305V / M306D / D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII, V158T / M298Q-FVII, V158D / E296V / M298Q-FVII K337A-FVII, M298Q-FVII, V158D / M298Q-FVII, L305V / K337A-FVII, V158D / E296V / M298Q / L305V-FVI I, V158D / E296V / M298A / F33I V158D / F33I / V158 FVII, K157A-FVII, E296V-FVII, E296V / M298Q-FVII, V158D / E296V-FVII V158D / M298K-FVII, and S336G-FVII, L305V / K337A-FVII, L305V / V158D-FVI-L155-V L305V / M298Q-FVII, L305V / V158T-FVII, L305V / K337A / V158T-FVII, L305V / K337A / M298Q-FVII, L305V / K337A / E296V-FVII L305V / K158 / V158-V158 / V158 , L305V / V158D / E296V-FVII, L305V / V158T / M298Q-FVII L305V / V158T / E296V-FVII, L305V / E296V / M298Q-FVII, L305V / V158D / E296V / M298Q-FV / V L305V / V158T / K337A / M298Q-FVII, L305V / V158T / E296V / K337A-FVII L305V / V158D / K337A / M298Q-FVII, L305V / V158D / E296V / K337A-FVI, L30V / FVI, L30 L305V / V158T / E296V / M298Q / K337A-FVII, S314E / K316H-FVII, S 314E / K316Q-FVII, S314E / L305V-FVII S314E / K337A-FVII, S314E / V158D-FVII, S314E / E296V-FVII, S314E / M298-FVII, S314E / V158T-FVII K35-FVII K3A FVII, K316H / V158D-FVII, K316H / E296V-FVII, K316H / M298Q-FVII K316H / V158T-FVII, K316Q / L305V-FVII, K316Q / K337A-FVII, K316Q / V158D-K316Q / V158D-F3 M298Q-FVII, K316Q / V158T-FVII, S314E / L305V / K337A-FVII, S314E / L305V / V158D-FVII S314E / L305V / E296V-FVII, S314E / L305V / M158-FVII / L305V / K337A / V158T-FVII, S314E / L305V / K337A / M298Q FVII, S314E / L305V / K337A / E296V-FVII, S314E / L305V / K337A / V158D-FVII S314E / L30-V3 / V158D / E296V-FVII, S314E / L305V / V158T / M298Q-FVII S314E / L305V / V158T / E296V-FVII, S314E / L305V / E296V / L305V / 6V / FVI / S314E / L305V / 15 / V158T / E296V / M298Q-FVII, S314E / L305V / V158T / K337A / M298Q-FVII S314E / L305V / V158T / E296V / K337A-FVII / S304V / V158D / K337A / S331 K337A-FVII, S314E / L305V / V158D / E296V / M298Q / K337A-FVII S314E / L305V / V158T / E296 V / M298Q / K337A-FVII, K316H / L305V / K337A-FVII, K316H / L305V / V158D-FVII K316H / L305V / E296V-FVII, K316H / L305V / M298Q-FVII, K316H / 30V / L3 / K337A / V158T FVII, K316H / L305V / K337A / M298Q-FVII, K316H / L305V / K337A / E296V-FVII, K316H / L305V / K337A / V308V / K15 / V158 / V158 / E296V-FVII, K316H / L305V / V158T / M298Q-FVII K316H / L305V / V158T / E296V-FVII, K316H / L305V / E296V / M298V / V158D / 815 KV / V158D / V158D / V158D / E296V / M298Q-FVII, K316H / L305V / V158T / K337A / M298Q-FVII K316H / L305V / V158T / E296V / K337A FVII, K316H / L305V / V158D / K337A / M293V-F15 , K316H / L305V / V158D / E296V / M298Q / K337A-FVII K316H / L305V / V158T / E296V / M298Q / K337A-FVII, K316Q / L305V / K337A-FVII, K316Q / L305V / F30I , K316Q / L305V / M298Q-FVII, K316Q / L305V / V158T-FVII, K316Q / L305V / K337A / V158T-FVII, K316Q / L305V / K337A / M298Q-FVII, K316Q / L305V / K30V / K306 / K337A / V158D-FVII K316Q / L305V / V158D / M298Q-FVII, K316Q / L305V / V158D / E296V-FVII, K316Q / L305V / V158T / M298Q-FVII K316Q / L305V / V158T / E296V-FVI I, K316Q / L305V / E296V / M298Q-FVI I, K316Q / L305V / V158D / E296V / M298Q-FV / M298Q-FV M298Q-FVII, K316Q / L305V / V158T / K337A / M298Q-FVII K316Q / L305V / V158T / E296V / K337A-FVI I, K316Q / L305V / V158D / K297A / M298Q-FVII K316 / K316Q / L305V / V158D / E296V / M298Q / K337A-FVII K316Q / L305V / V158T / E296V / M298Q / K337A-FVII, F374Y / K337A-FVII, F374Y / V158D-F37, F374-FVII, F374-FVII, F374 F374Y / V158T-FVII, F374Y / S314E-FVII, F374Y / L305V-FVII, F374Y / L305V / K337A-FVII F374Y / L305V / V158D-FVII / L305V / E296V-FVII, F30I / F30 L305V / V158T-FVII F374Y / L305V / S314E-FVII, F374Y / K337A / S314E-FVII, F374Y / K337A / V158T-FVII, F374Y / K297I / K377-F37I-K337A-K337A , F374Y / V158D / S314E-FVII, F374Y / V158D / M298Q-FVII F374Y / V158D / E296V-FVII F374Y / V158T / S314E-FVII, F374Y / V158T / M298Q-F37Y-F374 S314E-FVII F374Y / S314E / M298Q-FVII, F374Y / E296V / M298Q-FVII, F374Y / L305V / K33 7A / V158D FVII, F374Y / L305V / K337A / E296V-FVII, F374Y / L305V / K337A / M298Q-FVII, F374Y / L305V / K337A / V158T-FVI I F374Y / L305V / K337A / F337A / K337A / F334A / E296V-FVI I, F374Y / L305V / Vl58D / M298Q-FVII F374Y / L305V / V158D / S314E-FVII, F374Y / L305V / E296V / M298V-E376V / F294V / F294V / E296V / F294V / E296V / E296V / F294 / S314E-FVII, F374Y / L305V / M298Q / V158T-FVI I, F374Y / L305V / M298Q / S314E-FVII F374Y / L305V / V158T / S314E-FVII, F374Y / K337A / S314E /15 / V3 / M298Q-FVII F374Y / K337A / S314E / E296V-FVII, F374Y / K337A / S314E / V158D-FVII, F374Y / K337A / V158T / M298Q-E47 / F154T / V158T / E15 -FVII, F374Y / K337A / M298Q / V158D-FVII F374Y / K337A / E296V / V158D-FVII, F374Y / V158D / S314E / M298Q-FVII, F374Y / V158D / S314E / E296V-FVII F374Y / V158D / M298Q / E296V-F37, F37 / F37 / V158T / S314E / M298Q-FVII F374Y / V158T / M298Q / E296V-FVII, F374Y / E296V / S314E / M298Q-FVII, F374Y / L305V / M298A / 5314E-FVI F374 / E337A / 5314E-FVII F374 , F374Y / E296V / M298Q / K337A / 5314E-FVII F374Y / L305V / E296V / M298Q / K337A-FVII, F374Y / L305V / E296V / M298Q / V158D / F7I / E15 / E296V / M298Q / S314E-FVII F374Y / L305V / V158D / K337A / S314E-FVII, F374Y / V158D / M298Q / K337A / S314E-FVII / V158D / E296V / K337A / S14V / K337A / S33 / M298Q-FVII F374Y / L305V / V158D / M298Q / K337A-FVII, F374Y / L305V / V158D / E296V / K337A-FVII F374Y / L305V / V158D / M298Q / S314E-FV5, F37 / FVI, F37 F374Y / V158T / E296V / M298Q / K337A-FVII, F374Y / V158T / E296V / M298Q / S314E-FVII F374Y / L305V / V158T / K337A / S314E-FVII, F374Y / V158T / F294 / 715 E296V / K337A / S314E-FVII, F374Y / L305V / V158T / E296V / M298Q-FVII F374Y / L305V / V158T / M298Q / K337A-FVII, F374Y / L305V / V158T / E296V / K337A-FVII F374Y / L305V / V158T / M298Q / S314E-FVII, F374Y / L305V / V158T / E296V / S314E-V3E8V / S314E / S314E-FVII, F374Y / V158D / E296V / M298Q / K337A / S314E-FVII F374Y / L305V / V158D / E296V / M298Q / S314E-FVII / L295V / E296V / M298Q3 / 15V / E296V / M298V / M298Q / K337A / V158T-FVII, F374Y / L305V / E296V / K337A / V158T / S314E-FVII F374Y / L305V / M298Q / K337A / V158T / L305V / F304V / F30I / F30I / F304 / L305V / V158D / E296V / K337A / S314E-FVII, F374Y / L305V / V158D / M298Q / K337A / S314E-FVII F374Y / L305V / E296V / E338A / V158T / S314E / V158T / S314E M298Q / K337A / S314E-FVII, S52A-Factor VII, S60A-Factor VII; R152E-Factor VII S344A-Factor VII, T106N-FVII, K143N / N145T-FVII, V253N-FVII, R290N / A292T-FVII, G291N-FVII, R315N / V317T-FVII, K143N / N145T / R315- and FVII having substitutions, additions or deletions in the amino addition sequence from 233Thr to 240Asn; FVII having substitutions, additions or deletions in the amino acid sequence of 304Arg to 329Cys; and FVII having substitutions, additions or deletions in the amino acid sequence from 153Ile to 223Arg.

[0040] Variants of factor VII that have substantially the same or improved biological activity relative to wild-type factor VI I a comprise those that exhibit at least about 25%, such as, e.g. eg, at least about 50%, at least about 75%, at least about 90%, at least about 120%, at least about 130%, or at least about 150% of factor specific activity Wild type VIIa that has been produced in the same cell type, when tested in one or more coagulation assays, proteolysis assay, or TF binding assay as described above.

[0041] The biological activity of factor VIIa in blood coagulation derives from its ability to (i) bind to tissue factor (TF) and (ii) catalyze the proteolytic breakdown of factor IX or factor X to produce factor IX or X activated (factor IXa or Xa, respectively). For purposes of the invention, the biological activity of factor VIIa can be quantified by measuring the ability of a preparation to promote blood clotting using factor VII deficient plasma and thromboplastin, as described, e.g. eg, in US patent no. 5,997,864. In this assay, biological activity is expressed as the reduction in clotting time relative to a control sample and is converted to "factor VII units" by comparison with a pooled human serum level containing 1 unit / ml of factor VII activity. Alternatively, the biological activity of factor VIIa can be quantified by (i) measuring the ability of factor VIIa to produce Factor Xa in a system comprising TF embedded in a lipid membrane and factor X. (Persson et al., J Biol. Chem. 272: 19919-19924, 1997); (ii) measurement of factor X hydrolysis in an aqueous system (see, “General Methods" below); (iii) measurement of its physical binding to TF using an instrument based on surface plasmon resonance (Persson, FEBS Letts. 413: 359-363, 1997) (iv) measurement of hydrolysis of a synthetic substrate (see, "General Methods" below); and (v) measurement of thrombin generation in a TF-independent system in vitro (see "General methods" below).

O-linked glycosylation

[0042] In practicing the present invention, the oligosaccharide pattern can be determined using any method known in the art, including, without limitation: high performance liquid chromatography (HPLC); capillary electrophoresis (CE); nuclear magnetic resonance (NMR); mass spectrometry (MS) using ionization techniques such as fast atom bombardment, electrospray, or matrix assisted laser desorption (MALDI); gas chromatography (GC); and exoglycosidase treatment in conjunction with anion exchange (AIE) -HPLC, size exclusion chromatography (SEC), or MS. see, p. eg, Weber et al., Anal. Biochem. 225: 135 (1995); Klausen et al., J. Chromatog. 718: 195 (1995); Morris et al., In Mass Spectrometry of Biological Materials, McEwen et al., Eds., Marcel Dekker, (1990), pp. 137-167; Conboy et al., Biol. Mass Spectrom. 21: 397, 1992; Hellerqvist, Meth. Enzymol. 193: 554 (1990); Sutton et al., Anal. Biochem.

318: 34 (1994); Harvey et al., Organic Mass Spectrometry 29: 752 (1994). A method of performing O-glycosylation of small peptides is described in WO A 9518232.

[0043] The relative content of O-glycoforms can be determined, for example, by mapping tryptic peptides. Briefly, factor VII polypeptides are digested with trypsin and polypeptides containing the O-glycosylation site are separated according to glycan structure by RP-HPLC chromatography, mass spectrometry, or other suitable analytical separation technique. If necessary to obtain adequate separation, the factor ^v I ⁱ polypeptide can be reduced and alkylated before trypsin digestion, and the O-glycosylation site-containing polypeptide chain is purified by, e.g. ex. RP-HPLC chromatography. The purified factor VII polypeptide is then subjected to tryptic digestion followed by analysis as described above.

Methods for producing factor VII polypeptide preparations having a predetermined pattern of O-linked oligosaccharides

[0044] The origin of the acceptor factor VII polypeptide is not a critical aspect of the invention. Typically, the factor VII polypeptide will be expressed in a cultured prokaryotic cell or a eukaryotic cell such as a mammalian, insect, yeast, fungal, or plant cell. The protein, however, can also be isolated from a natural source such as plasma, serum or blood. The factor VII polypeptide can be either a full length protein or a fragment.

[0045] The invention provides compositions that include human factor VII polypeptide species that have a substantially uniform glycosylation pattern. The methods are useful for remodeling or altering the pattern of glycosylation present in a factor VII polypeptide in its initial expression. Thus, the methods of the invention provide a practical means for large-scale preparation of glycoforms having pre-selected or predetermined uniform derivatization patterns. The methods are particularly well suited for modification of therapeutic peptides, including but not limited to, factor VII polypeptides that are incompletely glycosylated during production in cell culture cells or transgenic animals. However, the compositions of the invention can also be prepared by purification from natural sources, such as plasma, serum or blood, or cell culture fluids and by isolation of the desired glycoforms thereof.

[0046] Factor VII polypeptides to be remodeled according to the invention are typically prepared by cell culture processes. Suitable host cells include, without limitation, human cells expressing an endogenous gene such as, e.g. eg, a factor VII gene. In these cells, the endogenous gene may be intact or it may have been modified in situ, or an external sequence of the endogenous gene may have been modified in situ to alter the expression of the endogenous factor VII polypeptide gene. Any human cell capable of expressing an endogenous factor VII polypeptide gene can be used. On the other hand, the included host cells are heterologous host cells programmed to express a factor VII polypeptide, from a recombinant gene. Host cells can be vertebrate, insect, or fungal cells. Preferably, the cells are mammalian cells capable of the entire spectrum of N-linked mammalian glycosylation; O-linked glycosylation; and y-carboxylation. See, p. Eg, US Patent Nos.

4,784,950. Preferred mammalian cell lines include CHO (ATCC CCL 61), COS-1 (ATCC CRL 1650), Kidney Hamster (BHK) and HEK293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36: 59-72, 1977). A preferred BHK cell line is the tk-ts13 BHK cell line (Waechter and Baserga, Proc.Natl.Acad.Sci.USA 79: 1106-1110, 1982), hereinafter referred to as BHK 570 cells. The BHK 570 cell line It is available from the American Type Culture Collection, 12301 Parklawn Dr., Rockville, MD 20852, under ATCC accession number CRL 10314. A tk-ts13 BHK cell line is also available from ATCC under accession number CRL 1632. In addition, a number of other cell lines can be used, including Rat Hep I (rat hepatoma; ATCC CRL 1600), Rat Hep II (rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), de human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1) and DUKX (CHO cell line) (Urlaub and Chasin, Proc. Natl. Acad. Sd. USA 77: 4216-4220, 1980)). (DUKX cells also referred to as CXB11 cells), and DG44 (CHO cell line) (Cell, 33: 405, 1983, and Somatic Cell and Molecular Genetics 12: 555, 1986). 3T3 cells, Namalwa cells, myelomas, and myeloma fusions with other cells are also useful. Suitable host cells include BHK 21 cells that have been adapted to grow in the absence of serum and have been programmed to express factor VII. The cells can be mutant or recombinant cells that express a qualitatively or quantitatively different spectrum of glycosylation enzymes (such as, eg, glycosyl transferases and / or glycosidases) than the cell type from which they were derived. Cells can also be programmed to express other heterologous peptides or proteins, including, eg. eg, truncated forms of factor VII. Host cells can also be CHO cells that have been programmed to co-express the factor VII polypeptide of interest (i.e., a factor VII or factor VII-related polypeptide) and another heterologous peptide or polypeptide such as, e.g. eg, a modifying enzyme or a factor VII fragment.

[0047] Methods: The present invention comprises methods of producing a preparation comprising a predetermined serine-linked glycoform model as described above. Methods for optimizing the glycoform distribution of a factor VII polypeptide are also described herein (see Figure 3). The described individual process steps can be applied in different combinations to obtain the desired glycoform pattern. Non-limiting reference examples are given below.

[0048] In one aspect, these methods are performed in the phases of:

(a) obtaining a preparation of a factor VII polypeptide containing a Cys-X1-Ser / Thr-X2-Pro-Cys motif and where said serine forms part of a Glc-O-Ser covalent bond of a cell in which prepare; p. eg, from a genetically engineered cell (cell culture) or by isolation of factor VII polypeptide from a natural source;

(b) contacting the preparation of the factor VII polypeptide with an activated donor of the desired fraction of the mono- or oligosaccharide and an enzyme suitable for transfer of the desired group of mono- or oligosaccharides under conditions appropriate for transfer of the group of mono- or or oligosaccharides from the donor fraction to the acceptor fraction, thereby producing the factor VII polypeptide with an altered glycosylation pattern.

[0049] In another aspect, these methods are carried out in the phases of:

(aa) obtaining a preparation of a factor VII polypeptide containing a Cys-X1-Ser / Thr-X2-Pro-Cys motif and where said serine forms part of a Glc-O-Ser covalent bond of a cell in which it is prepared; p. eg, from a genetically engineered cell (cell culture) or by isolation of factor VII polypeptide from a natural source;

(bb) contacting the preparation of the factor VII polypeptide with an enzyme suitable for the removal of the terminal group of mono- or oligosaccharides under appropriate conditions for the removal of said group of mono- or oligosaccharides, thereby producing the factor VII polypeptide with an altered model of glycosylation.

[0050] The methods described here may comprise a combination of steps (b) and (bb). The methods further comprise an isolation phase of the factor VII polypeptide with an altered pattern of glycosylation.

[0051] The methods described here may comprise another phase of:

[0052] Analysis of the structure of oligosaccharides linked to factor VII polypeptides to determine a glycoform pattern, and, optionally, repetition of steps (b) and / or (bb) until a desired glycoform pattern be got.

[0053] These methods may further comprise the step of subjecting preparations having predetermined glycoform patterns to at least one bioactivity test (including, eg, coagulation, factor X proteolysis, or TF binding) or other functionality ( such as, eg, pharmacokinetic profile or stability), and correlating particular glycoform patterns with particular bioactivity or functionality profiles to identify a desired glycoform pattern.

[0054] The desired glycoform model is a substantially uniform xylose-xylose-glucose-O-serine glycosylation: in this embodiment, the method ( METHOD A2) comprises the steps of:

(a) obtaining a preparation of a factor VII polypeptide containing a Cys-X1-Ser-X2-Pro-Cys motif and where said serine is located at position 52 and forms part of a Glc-O-Ser covalent bond; p. eg, from a genetically engineered cell (cell culture) or by isolation of factor VII polypeptide from a natural source;

(b) contacting the preparation obtained in step (a) with UDP-D-xylose: pD-glucoside α-1,3-D-xylosyltransferase and an activated donor of xylosyl under appropriate conditions to transfer a xylose residue from the fraction xylose donor to the acceptor fraction, thereby producing the factor VII polypeptide with an altered glycosylation pattern.

(c) contacting the preparation obtained in step (b) with a UDP-D-xylose: aD-xyloside a-1,3-D-xylosyltransferase and an activated donor of xylosyl under appropriate conditions to transfer a xylose residue from the xylose donor fraction to the acceptor fraction, thereby producing the factor VII polypeptide with an altered glycosylation pattern.

[0055] In one embodiment, the method further includes the isolation phase of the preparation obtained in step (b) before subjecting the preparation to step (c).

[0056] In one embodiment, the method further includes the isolation step of the factor VII polypeptide prepared in step (c) with an Xyl-Xyl-Glc-O-Ser glycosylation.

[0057] In one embodiment, the method further includes the analysis phase of the structure of the oligosaccharides linked to the factor VII polypeptides to determine a glycoform pattern, and, optionally, the repetition of phase (b) and / or step (c) until the desired glycoform model has been achieved.

[0058] The factor VII polypeptide exhibits substantially uniform Xyl-Xyl-Glc-O-Ser glycosylation; Being being the serine of the Cys-X1-Ser-X2-Pro-Cys motif contained (X1 and X2 independently being any amino acid residue).

[0059] In preferred embodiments, the preparation of the human factor FVII polypeptide is selected from the list of:

Factor VII polypeptides exhibiting substantially uniform Xyl-Xyl-Glc-O-Ser52 glycosylation, Factor VII polypeptides exhibiting substantially uniform Xyl-Glc-O-Ser52 glycosylation Variants of Factor VII exhibiting Xyl-Xyl-Glc-O- glycosylation Ser52 substantially uniform

[0060] It should be understood that oligosaccharides such as Xyl-Xyl- can also be transferred to the Glc-O-Ser / Thr acceptor fraction using a suitable transfer enzyme and an activated donor of Xyl-Xyl-.

[0061] Chromatographic method: Hydrophobic interaction chromatographic methods are described herein to produce a preparation comprising a predetermined serine-linked glycoform model as described above, and to purify an O-glycosylated factor VII polypeptide with a desired glycoform model of a composition comprising said factor VII polypeptide and factor VII polypeptides having unwanted glycoform patterns.

[0062] In one aspect, the method comprises the following steps:

(a) obtaining a preparation of a factor VII polypeptide containing a Cys-X1-Ser-X2-Pro-Cys motif and where said serine forms part of a Glc-O-Ser covalent bond of a cell in which it is prepared ; p. eg, from a genetically engineered cell (cell culture) or by isolation of factor VII polypeptide from a natural source;

(b) binding of the factor VII polypeptide to a hydrophobic interacting material using a solution comprising water, optionally a salt component, and optionally a buffer,

(c) optionally washing the hydrophobic interaction material using a solution comprising water, optionally a salt component, and optionally a buffer to elute contaminants from the hydrophobic interaction material;

(d) washing the hydrophobic interaction material using a solution comprising an organic modifier, water, optionally a salt component, and optionally a buffer, to a linear gradient or a phase gradient or isocratically in a salt component to separate the polypeptide factor VII with a desired glycoform model of the factor VII polypeptide that does not have the desired glycoform of the hydrophobic interacting material;

(e) collecting the fraction containing the factor VII polypeptide having the desired glycoform model.

[0063] The methods described above may further include the step of repeating steps (a) through (e) by subjecting the preparation obtained in step (e) to steps (a) through (e). This additional phase can be repeated more than once if deemed necessary.

[0064] It should be understood that the preparations described herein can also be prepared by a process comprising a combination of purification steps whereby factor VII polypeptide species having the desired glycosylation are captured from the cell culture fluid or natural source of origin and the enzymatic methods described above.

[0065] The methods described above may further comprise the step of subjecting preparations having predetermined glycoform patterns in at least one bioactivity test (including, eg, coagulation, factor X proteolysis, or TF binding) or other functionality (such as, eg, pharmacokinetic profile or stability), and correlating particular glycoform patterns with particular bioactivity or functionality profiles to identify a desired glycoform pattern.

[0066] Other enzyme treatments can be used in conjunction with the above methods to modify the N- or O-linked glycan oligosaccharide model of a preparation; Such treatments include, without limitation, treatment with one or more of sialidase (neuraminidase), galactosidase, fucosidase; galactosyl transferase, fucosyl transferase, and / or sialyltransferase, in a sequence and under conditions that achieve a desired modification in the distribution of oligosaccharide chains having particular terminal structures. Glycosyl transferases are commercially available from Calbiochem (La Jolla, CA) and glycosidases are commercially available from Glyko, Inc., (Novato, CA).

Factor VII polypeptide preparations

[0067] As used herein, a "factor VII polypeptide preparation" refers to a plurality of factor VII glycoforms that have been separated from the cell in which they were synthesized. The factor VII polypeptide preparation is a preparation of human factor VII polypeptides and includes forms activated, functionally related polypeptides such as e.g. eg, chemically modified variants and forms, which have been separated from the cell in which they were synthesized.

[0068] For example, as used herein, a "factor VII preparation" refers to a plurality of factor VII polypeptides, factor VI I to polypeptides, or factor VII related polypeptides, including chemically modified variants and forms. , which have been separated from the cell in which they were synthesized or isolated from the natural source.

[0069] Separation of factor VII polypeptides from their cell of origin can be accomplished by any method known in the art, including, without limitation, removal of cell culture medium containing the desired product of an adherent cell culture; centrifugation or filtration to remove non-adherent cells; and the like.

[0070] Optionally, the polypeptides can be further purified. Purification can be accomplished using any method known in the art, including, without limitation, affinity chromatography, such as, e.g. eg, in an anti-factor VII (see, eg, Wakabayashi et al., J. Biol. Chem. 261: 11097, 1986; and Thim et al., Biochem. 27: 7785, 1988); hydrophobic interaction chromatography; ion exchange chromatography; size exclusion chromatography; electrophoretic procedures (eg, preparatory isoelectric focusing (IEF), differential solubility (eg, ammonium sulfate precipitation), or extraction, and the like. See generally Scopes, Protein Purification, Springer Verlag, New York, 1982 and Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989. After purification, the preparation preferably contains less than about 10% by weight, more preferably less than about 5 % and most preferably less than about 1%, of unrelated proteins derived from the host cell.

[0071] Factor VII and factor VII-related polypeptides, respectively, can be activated by proteolytic cleavage, using factor XIIa or other proteases that have trypsin-like specificity, such as, e.g. eg, factor IXa, kallikrein, factor Xa, and thrombin. See, p. eg, Osterud et al., Biochem. 11: 2853 (1972); Thomas, US Patent No. 4,456,591; and Hedner et al., J. Clin. Invest. 71: 1836 (1983). Alternatively, factor VII can be activated by passing it through an ion exchange chromatography column, such as Mono Q® (Pharmacia) or the like. The resulting activated factor VII polypeptide can then be formulated and administered as described below.

Functional properties of factor VII polypeptide preparations

[0072] Preparations of factor VII polypeptides having predetermined oligosaccharide patterns described herein (including factor VII polypeptides, factor VII related polypeptides, exhibit improved functional properties relative to reference preparations. Improved functional properties may include, without limitation, a) physical properties such as e.g. eg, storage stability; b) pharmacokinetic properties such as e.g. eg, bioavailability and half-life; c) immunogenicity in humans, and d) biological activity, such as, e.g. eg, coagulating activity.

[0073] A reference preparation refers to a preparation comprising a polypeptide that is identical to that contained in the preparation of the invention with which it is compared (such as, eg, wild-type factor VII or a particular variant or chemically modified form) except to exhibit a different serine / threonine-linked glycosylation pattern.

[0074] The storage stability of a preparation of factor VII polypeptides can be evaluated by measuring (a) the time required during 20% of the bioactivity of a preparation to deteriorate when stored as a dry powder at 25 ° C and / or or (b) the time required for a doubling in the proportion of (eg, factor VIIa) aggregates of said factor VII polypeptide in the preparation.

[0075] In some embodiments, the preparations exhibit an increase of at least about 30%, preferably at least about 60%, and more preferably at least about 100%, in the time required for 20% bioactivity. to deteriorate in relation to the time required for the same phenomenon in a reference preparation, when both preparations are stored as dry powders at 25 ° C. Bioactivity measurements can be performed using any coagulation assay, proteolysis assay, TF binding assay, or TF independent thrombin generation assay.

[0076] In some embodiments, the preparations of the invention exhibit an increase of at least about 30%, preferably at least about 60%, and more preferably at least about 100%, in the time required for duplication of aggregates in relation to a reference preparation, when both preparations are stored as dry powders at 25 ° C. The content of aggregates can be determined according to methods known to the person skilled in the art, such as, for example, eg, gel permeation HPLC methods. For example, the aggregate content of factor VII is Determine by HPLC gel permeation on a Pak 300 SW protein column (7.5 x 300 mM) (Waters, 80013) as follows. The column is equilibrated with eluent A (0.2M ammonium sulfate, 5 % isopropanol, pH adjusted to 2.5 with the addition of phosphorus, and the pH is then adjusted to 7.0 with triethylamine), after the which 25 pg of sample is applied to the column. Elution is with eluent A at a flow rate of 0.5 ml / min for 30 min, and detection is achieved by measuring the absorbance at 215 nm. The aggregate content is calculated as the area of the maximum value of factor VII aggregates / total area of maximum factor VII values (monomer and aggregates).

[0077] "Bioavailability" refers to the proportion of an administered dose of a preparation of factor VII polypeptides (eg, factor VII or related to factor VII) that can be detected in plasma at predetermined times after the administration. Typically, bioavailability is measured in experimental animals by administering a dose of between about 25-250 pg / kg of the preparation; obtaining plasma samples at predetermined times after administration; and determining the content of glycosylated factor VII (eg, factor VII or factor VII related) polypeptides in the samples using one or more of a coagulation assay (or any bioassay), an immunoassay, or an equivalent. . Data is typically displayed graphically as factor VII v polypeptide. time and bioavailability is expressed as the area under the curve (AUC). The relative bioavailability of a test preparation refers to the ratio between the AUC of the test preparation and that of the reference preparation.

[0078] In some embodiments, the preparations exhibit a relative bioavailability of at least about 110%, preferably at least about 120%, more preferably at least about 130%, and most preferably at least about 140% of the bioavailability of a reference preparation. Bioavailability can be measured in any mammalian species, preferably dogs, and the predetermined times used to calculate AUC can comprise different increments of 10 min-8 h.

[0079] "Half-life" refers to the time required for plasma concentration of (eg, factor VII polypeptides from factor VII-related polypeptides) to factor VII polypeptide to halve a particular value of this value. Half-life can be determined using the same procedure for bioavailability. In some embodiments, the preparations exhibit an increase in half-life of at least about 0.25 h, preferably at least about 0.5 h, more preferably at least about 1 h, and most preferably at least about 2 h , in relation to the half-life of a reference preparation.

[0080] "Immunogenicity" of a preparation refers to the ability of the preparation, when administered to a human, to elicit a deleterious immune response, be it humoral, cellular, or both. It is known that factor VIIa polypeptides and factor VIIa related polypeptides do not elicit detectable immune responses in humans. However, in any human subpopulation, there may be individuals that exhibit sensitivity to particular administered proteins. Immunogenicity can be measured by quantifying the presence of anti-factor VII antibodies and / or factor VII-responsive T cells in a sensitive individual, using standard methods known in the art. In some embodiments, the preparations exhibit a reduction in immunogenicity in a sensitive individual of at least about 10%, preferably at least about 25%, more preferably at least about 40%, and most preferably at least about 50%, relative to the immunogenicity for this individual of a reference preparation.

Pharmaceutical compositions and methods of use

[0081] The preparations can be used to treat any syndrome that responds to the relevant glycoprotein. Factor VII, FIX and FX-responsive syndromes, respectively, include syndromes such as, e.g. eg, bleeding disorders, including, without limitation, those caused by clotting factor deficiencies (eg, hemophilia A and B or clotting factor XI or VII deficiency); from thrombocytopenia or von Willebrand disease, or from clotting factor inhibitors, or excessive bleeding from any cause. The preparations can also be administered to patients in association with surgery or other trauma or to patients receiving anticoagulant therapy.

[0082] Pharmaceutical compositions comprising factor VII and factor VII related preparations herein are primarily intended for parenteral administration for prophylactic and / or therapeutic treatment. Preferably, the pharmaceutical compositions are administered parenterally, that is, intravenously, subcutaneously, or intramuscularly. They can be administered by continuous or pulsatile infusion.

[0083] Pharmaceutical compositions or formulations according to the invention comprise a preparation in combination with, preferably dissolved in, a pharmaceutically acceptable carrier, preferably an aqueous carrier or diluent. A variety of aqueous carriers can be used, such as water, water buffered, 0.4% saline, 0.3% glycine and the like. The preparations of the invention can also be formulated into liposome preparations for delivery or targeting to wound sites. Liposome preparations are generally described in, eg. eg, US Patent Nos. 4,837,028, 4,501,728, and 4,975,282. The compositions can be sterilized by conventional, well known sterilization techniques. The resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.

[0084] The compositions may contain pharmaceutically acceptable auxiliaries or adjuvants, including, without limitation, pH adjusting agents and buffering agents and / or tonicity adjusting agents, such as, for example, sodium acetate, sodium lactate, chloride sodium, potassium chloride, calcium chloride, etc.

[0085] The concentration of factor VII polypeptides or factor VII related polypeptides in these formulations can vary widely, ie, from less than about 0.5% by weight, typically to or at least about 1% by weight to as much as 15 or 20% by weight and will be selected mainly for fluid volumes, viscosities, etc., according to the particular mode of administration selected.

Thus, a typical pharmaceutical composition for intravenous infusion could be formed to contain 250 ml of sterile Ringer's solution and 10 mg of the preparation. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA (1990).

[0087] The compositions of the present invention can be administered for prophylactic and / or therapeutic treatments. In therapeutic uses, the compositions are administered to a subject already suffering from a disease, as described above, in an amount sufficient to cure, alleviate, or partially stop the disease and its complications. A suitable amount to accomplish this is defined as "therapeutically effective amount". Effective amounts for each purpose will depend on the severity of the disease or injury as well as the weight and general condition of the subject. In general, however, the effective amount will vary from about 0.05 mg to about 500 mg of the preparation per day for a 70 kg subject, with dosages of about 1.0 mg to about 200 mg of the preparation being used more. commonly daily. It will be understood that determination of an appropriate dosage can be accomplished using routine experimentation, by constructing a matrix of values, and by testing different points on the matrix.

[0088] Local delivery of the preparations, such as, for example, topical application, can be carried out, eg. Eg, by spraying, perfusion, spiral double-balloon catheters, incorporated into vascular or spiral grafts, hydrogels used to cover balloon catheters, or other well-established methods. Either way, the pharmaceutical compositions should provide an amount of the preparation sufficient to effectively treat the subject.

[0089] The pharmaceutical compositions of the invention may further comprise other bioactive agents, such as, e.g. eg, coagulants or anticoagulants not related to factor VII.

EXPERIMENTAL

General methods

Test ^to -xilosidasa

[0090] Assays for a-xylosidase are performed in an appropriate buffer, eg. ex. 50 mM sodium acetate, pH 4.5, containing a suitable substrate, e.g. ex. the O-glycopeptides that can be obtained from the O-glycopeptide map of the relevant glycoprotein (eg rFVIIa). The reaction stops after an appropriate time that can be experimentally determined, eg. ex. the addition of trifluoroacetic acid, and the test mixtures are analyzed by HPLC.

Test ^to -xilosiltransferasa

[0091] Assays for α-cytosyltransferase are performed in an appropriate buffer, eg. ex. 10 mM Hepes, pH 7.2, 0.1% Triton X-100, 0.5 mM UDP-xylose (Sigma U5875), containing a suitable substrate, e.g. ex. the O-glycopeptides that can be obtained from the O-glycopeptide map of the relevant glycoprotein (eg rFVIIa) or the pyridyl-amino oligosaccharides prepared as described in Minamida et al. (Minamida et.al., Detection of UDP-D-xylose: aD-xyloside a1- 3xylosyltransferase activity in human hepatoma cell line Hep2. J.

Biochem. 120 1002-1006, 1996). The reaction stops after an appropriate time, which can be determined experimentally, by e.g. ex. the addition of trifluoroacetic acid, and the test mixtures are analyzed by HPLC.

[0092] Assays for α-xylosidase and α-xylosyltransferase are optimized for time and, optionally, for temperature and pH.

O-glucosyltransferase assay.

[0093] O-Glucosyltransferase assays are performed, eg. eg, as described by Shao et al. (Glycobiology 12 (11) 763-7702002).

Factor VII tests

[0094] A suitable assay for testing for factor VIIa activity and thus the selection of suitable variants of factor VIIa can be performed as a simple preliminary in vitro test . The assay is also suitable for selecting suitable variants of factor VIIa.

In vitro hydrolysis test

[0095] Variants of native factor VIla (wild type) and factor VIIa (both hereinafter referred to as "factors VIIa") can be evaluated for specific activities. They can also be evaluated in parallel to directly compare your specific activities. The assay is performed on a microtiter plate (MaxiSorp, Nunc, Denmark). Chromogenic substrate D-Ile-Pro-Arg-p-nitroanilide (S-2288, Chromogenix, Sweden), final concentration of 1 mM is added to factor VIla (final concentration of 100 nM) in 50 mM Hepes, pH 7.4 , containing 0.1 M NaCl, 5 mM CaCl ² and 1 mg / ml of bovine serum albumin. Absorbance at 405 nm is continuously measured on a spectraMax ™ 340 plate reader (Molecular Devices, USA). Absorbance developed during a 20 minute incubation, after subtraction of absorbance in a blank well containing no enzyme, is used to calculate the ratio between the activities of variant factor VIIa and wild type factor VIIa:

Proportion = (A ^{4 0 5} nm variant of factor VIIa) / (A ^{4 0 5} nm wild type factor VIIa).

[0096] Based on this, factor VIIa variants with activity comparable to or greater than native factor VIIa can be identified, such as, for example, variants where the ratio of variant activity to native factor VII activity (FVII wild type) is around, against greater than 1.0.

[0097] The activity of factor VIIa or variants of factor VIIa can also be measured using a physiological substrate such as factor X, suitably at a concentration of 100-1000 nM, where the generated Factor Xa is measured after the addition of a suitable chromogenic substrate (eg S-2765). Furthermore, the activity assay can be performed at physiological temperature.

In Vitro Proteolysis Assay

[0098] Native factor VIIa (wild-type) and factor VIIa variant (both hereinafter referred to as "factor VII") are tested in parallel to directly compare their specific activities. The assay is performed on a microtiter plate (MaxiSorp, Nunc, Denmark). Factor VIIa (10 nM) and factor X (0.8 microM) in 100 microL 50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 5 mM CaCl2 and 1 mg / ml serum albumin bovine, incubate for 15 min. The division of factor X is then stopped by the addition of 50mL 50mM Hepes, pH 7.4, containing 0.1M NaCl, 20mM EDTA and 1mg / ml bovine serum albumin. The amount of Factor Xa generated is measured by the addition of the chromogenic substrate Z-D-Arg-Gly-Arg-P-nitroanilide (S-2765, Chromogenix, Sweden), final concentration of 0.5mM. Absorbance at 405 nm is continuously measured on a SpectraMax ™ 340 plate reader (Molecular Devices, USA). The absorbance developed for 10 minutes, after the absorbance subtraction in a blank well containing no FVIIa, is used to calculate the ratio between the proteolytic activities of the variant factor VIIa and wild type factor VIIa:

Proportion = (A ^{4 0 5} nm variant of factor VIIa) / (A405nm factor VIIa wild type).

[0099] Based on this, factor VIIa variants with activity comparable to or greater than native factor VIIa can be identified, such as, for example, variants where the ratio of variant activity to native factor VII activity ( wild-type FVII) is around 1, against greater than 1.0.

Thrombin generation assay:

[0100] The ability of factor VII or factor VII related polypeptides (eg, variants) to generate thrombin can be measured in an assay comprising all relevant coagulation factors and inhibitors at physiological concentrations and activated platelets (such as is described on p. 543 in Monroe et al. (1997) Brit. J. Haematol. 99, 542-547 which is incorporated herein by reference).

Clot assays.

First generation trial

[0101] The activity of factor VII polypeptides can also be measured using a one-step clot assay essentially as described in WO 92/15686 or US 5,997,864. Briefly, the sample to be evaluated is diluted in 50 mM Tris (pH 7.5), 0.1% BSA and 100 µl is incubated with 100 µl of factor VII deficient plasma and 200 µl of thromboplastin C containing 10 mM Ca2 +. Clotting times are measured and compared to a standard curve using a reference standard or a group of citrated normal human plasma in serial dilutions.

Second generation test:

[0102] Essentially the same, except that human recombinant tissue factor is used instead for thromboplastin C.

Factor IX test

Test for factor IX activity:

[0103] Assays suitable for testing for factor IX activity, and thus providing means for selecting suitable factor IX variants for use in the present invention, may be performed as simple in vitro tests as described, eg, in Wagenvoord et al., Haemostasis 1990,20 (5): 276-88 [0104] The biological activity of factor IX can also be quantified by measuring the ability of a preparation to correct the coagulation time of factor IX-deficient plasma, p . Eg, as described in Nilsson et al., 1959. (Nilsson IM, Blombaeck M, Thilen A, von Francken I., Carriers of haemophilia A-A laboratory study, Acta Med Scan 1959, 165: 357). In this assay, biological activity is expressed as units / ml plasma (1 unit corresponds to the amount of FIX present in normal pooled plasma.

Examples

[0105] The following examples are intended as non-limiting illustrations of the present invention.

Example 1

Preparation of ^a -xilosidasa by extraction and purification

[0106] The enzyme, a-xylosidase, can be prepared from different sources, eg. ex. of plant material as described by Monroe et al. (Plant Physiology and Biochemistry 41: 877-885 (2003)). For example, plant tissues from e.g. ex. Arabidopsis thaliana are ground in a mortar and pestle with quartz sand in two volumes of Buffer A (40 mM Hepes, pH 7.0, 1 M NaCl), and the filtered extract is centrifuged at 15000 x g for 15 min. Ammonium sulfate is added for example at 80% saturation. The precipitated proteins are collected by centrifugation at 15000 xg for 15 min and are redissolved in buffer A. The a-xylosidase is purified by chromatography, for example on a Concanavalin A-Sepharose column, on an anion exchange column and / or in other chromatographic columns known to the person skilled in the art. Fractions are collected during elution and fractions containing the enzyme α-xylosidase are identified using the α-xylosidase assay.

Example 2

Preparation of ^a -xilosidasa by cloning and expression in E. coli and purification

[0107] Genes encoding α-xylosidases, which can hydrolyze alpha-xylosidic linkages, have been cloned and previously characterized, and genes showing significant homology with characterized α-xylosidases have been annotated in the genomes of various prokaryotic and eukaryotic organisms. The gene sequences are available in databases such as SWISS-PROT or NCBI and can be PCR amplified from genomic DNA of the respective organisms. Several candidates were chosen for cloning and expression in E. coli after searching protein databases for the presence of α-xylosidase proteins. The following candidate genes were selected based on the existing annotation in the databases (A), published previous characterization (P) or based on known homology to α-xylosidases (H): gene tm0308 (Thermotoga maritima: A); gene bt3085 (2139 bp) and gene bt3659 (2475 bp) (Bacteroides thetaiotaomicron: A); gene bf0551 (2238 bp) and gene bf1247 (2538 bp) (Bacteroides fragilis: A); gene b102681 (2310 bp) (Bacillus licheniformis: H); bh gene 1905 (2328 bp) (Badllus halodurans: H), gene xylS (2196 bp) (Sulfolobus solfataricus: P); yicI gene (2319 bp) (Escherichia coli: P)

Strategy for cloning and expression in E. coli ^to -xilosidasas

[0108] SignaIP software (Bendtsen, J.D. et al.J. Mol. Biol., 340: 783-795, 2004) is used to assess whether a signal peptide is potentially present at the N-terminus of candidate enzymes. BF0551; BF1247; BT3085; BT3659 are supposedly secreted as indicated by a strong prediction of a signal I peptidase cleavage site. A methionine codon encoding a start methionine is included in front of the first amino acid after the predicted cleavage site.

[0109] Purified genomic DNA from Bacteroides thetaiotaomicron (ATCC 29148D), Bacteroides fragilis (ATCC 25285D), Bacillus haludurans (ATCC 21591 D & BAA-125D), Sulfolobus solfataricus (ATCC 35092D), Thermotoga maritima (ATCC 4358D) Collection. In the case of E. coli (derived from strain K-12) and Bacillus licheniformis (ATCC 28450), genomic DNA is prepared from bacterial cells grown overnight in LB medium using the DNeasy tissue kit (Qiagen ) according to the manufacturing instructions.

[0110] Forward and reverse primers for PCR amplification are designed with an extension at the 5 'ends comprising the cleavage sites of the restriction enzymes NdeI (or XbaI) and XmaI, respectively. PCR is performed using the following condition: 1) 95 ° C for 3 min: denaturation, 2) 94 ° C for 30 sec: denaturation, 3) 55 ° C or 60 ° C for 30 sec: alignment, 4) 72 ° C for 2 min: elongation. Phase 2-4 is repeated for 15 cycles. PCR products are separated on 1% ethidium bromide agarose gels and bands showing the correct predicted sizes are cut from the gels and purified using the GFX DNA Purification Kit (Amersham Pharmacia). The PCR purified products are cloned into the pCR2.1TOPO vector according to the manufacturer's instructions (Invitrogen). Clones showing the correct restriction enzyme cleavage profile are sorted to assess DNA sequence. The insert representing the a-xylosidase genes are released from the vector pCR2.1TOPO using the relevant restriction enzymes. An E. coli pET11a expression vector (Novagen) containing an NdeI (and XbaI) and XmaI site is cleaved with the relevant restriction enzymes and the part of the vector is purified as described for the PCR products. Vector and inserts are ligated together using the Rapid Ligation Kit (Roche) according to the manufacturer's instructions.

[0111] The ligation products are transformed into E. coli TOP10 cells (Invitrogen) by chemical transformation or heat shock methods known to those skilled in the art. Cells are plated onto LB / Ampicillin (Amp) culture plates overnight. Individual colonies are selected from the plates and grown overnight in LB / Amp medium. Purified pET plasmids from each colony are screened for the presence of correct inserts using restriction cleaving enzymes and evaluating the sizes of the released inserts.

[0112] Rosetta DE3 from E. Coli (Novagen) is transformed with pET plasmids containing the axilosidase genes and plated on chloramphenicol (Cam) / Amp LB. Cells from plates left overnight are resuspended in Cam / Amp LB liquid medium and diluted to OD ⁶⁰⁰ = 0.1. The cells of the liquid medium propagate up to OD ⁶⁰⁰ = 0.4-0.8. The cells are then equilibrated at a temperature of 18 ° C for 30 min. and protein induction is induced with 0.5 mM IPTG overnight at 18 ° C. The cells are harvested and the granules are resuspended in a buffer (eg, 25mM Tris HCI pH 7 or 10mM potassium phosphate buffer pH 7) at a cell density corresponding to OD ⁶⁰⁰ = ~ 10. Cells are sonicated on ice for 3-7 times 15-30 sec with 30 sec interruptions on ice. Cell debris is removed by centrifugation and supernatants are assayed for activity.

Test for activity -xilosidasa

[0113] Supernatants resulting from sonication are evaluated in p-nitrophenyl aD xylopyranoside (Sigma) for the presence of a-xylosidase activity. Crude enzyme is incubated with 5mM p-nitrophenyl aD xylopyranoside at 37 ° C for 1-2 hours in a buffer (eg, 10mM potassium buffer pH 7 or 25mM Tris HCI buffer pH 7). Crude enzymes are also tested on a fragment of human FVII comprising Xyl-Xyl-Glc-O-Ser52 glycosylation (peptide fragment consisting of amino acid residues 39-62 of FVII) to assess whether the enzyme can split alpha bonds 1.3 xylosidics. Incubation with peptide is carried out for 3 hours or overnight at 37 ° C. Peptide samples incubated with or without α-xylosidase are then evaluated by MALDI MS directly after incubation to assess whether the enzyme can remove zero, one, or two xylose sugars from the glycopeptide.

Purification of ^a -xilosidasa

[0114] A partial purification of the expressed α-xylosidase is performed prior to incubation with rFVII. Supernatants (from approximately 20-50 ml of cell culture) obtained after cell disruption in a suitable buffer (eg, a 10mM phosphate buffer pH 7). In the case of enzymes that come from thermophiles (for example tm0308; BH1905, XylS), the supernatants are also heated at 50-70 ° C for 30 min, chilled on ice for 10 min and the precipitate is removed by centrifugation for 15 min at 15,000 G to remove thermolabile E. coli contaminants .

[0115] The supernatants are sterile filtered and applied to a 1 ml DEAE FF column (Amersham Pharmacia). Purification is performed with the AKTA explorer FPLC system (Amersham Pharmacia) with the following buffers: buffer A: 25mM sodium phosphate pH 7, buffer B: 25mM sodium phosphate pH 7 and 1M NaCl. After loading the application, the unbound sample is washed with buffer A for 5 CV. A 0-100% gradient of buffer B is used over 20 CV during which the target protein is eluted in fractions. After purification, the fractions comprising the main maximum value in the resulting chromatogram are evaluated by incubation in p-nitrophenyl aD xylopyranoside or by SDS PAGE. Fractions containing α-xylosidase activity are diluted in 20mM Tris HCl pH 7 buffer, 2mM CaCl ² and concentrated on 50,000 MWCO Vivaspin 20 columns (Vivascience) by centrifugation at 2900 rpm.

O-glycoforms of rFVIIa exclusively with serine glucose 52

[0116] O-glycoforms of rFVIIa exclusively with glucose in serine 52 are obtained from incubation of rFVIIa in an appropriate buffer, eg. ex. glycyl glycine or 20mM Tris HCl pH 7.0, 2mM CaCl2, with purified α-xylosidase for an appropriate time, which can be determined experimentally. Mass spectra visualizing deglycosylation are obtained from analysis of rFVII a-xylosidase ESI-MS (Q-STAR) incubations.

[0117] The resulting glycan remodeled rFVIIa is purified from the enzyme α-xylosidase for example by anion exchange chromatography or gel filtration or suitable combinations. The purity of the prepared rFVIIa O-glycoform is verified by the rFVIIa O-glycopeptide map.

Example 3

Preparation of ^a -xilosidasa by cloning and expression of the putative gene -xilosidasa ^to T. maritima (tm0308) in E. coli and purification

[0118] The above mentioned strategy (see example 2) was followed all the way to a conclusion for tm0308. The putative T. maritime a-xylosidase gene (tm0308) was amplified by PCR and cloned into an E. coli pET11a vector . Soluble tm0308 could be obtained after expression in an E. coli Rosetta expression strain (DE3) and evaluation of a crude preparation of TM0308 in an aD xylopyranoside pnitrophenyl clearly indicated α-xylosidase activity. The α-xylosidase was partially purified using DEAE FF chromatography followed by ascending concentration by ultrafiltration. The partially purified enzyme was incubated with FVII in a 25mM Tris pH 7 buffer, 2mM CaCl ² at different enzyme / FVII ratios for 3 hours at 50 ° C or overnight at 37 ° C. Controls with identical compositions of axilosidase and rFVII, to which synthetic substrate was added, showed that the enzyme was active under these conditions and it was possible to visualize FVII with and without xylosidase treatment in SDS gels and by ESI-MS. However, no significant elimination of xylose sugars bound to Glc-O-Ser52 could be detected in this first experiment. In contrast, the removal of xylose from an alkylated, reduced purified FVIIa peptide comprising Xyl-Xyl-Glc-O-Ser52 was observed.

Example 4

Preparation of ^a -xilosidasa for cloning and expression in E. coli

[0119] The following constructs have been cloned into the pET expression vectors according to the strategy described in Example 2: gene bI02681 (2310 bp) (Bacillus licheniformis: H); b11905 gene (2328 bp) (Bacillus halodurans: H), xylS gene (2196 bp) (Sulfolobus solfataricus: P); yicI gene (2319 bp) (Escherichia coli: P).

Constructs will be expressed in Rosetta, isolated, purified, and evaluated for α-xylosidase activity according to the strategy described above.

Each α-xylosidase will be incubated with rFVIIa in an appropriate buffer, e.g. ex. glycyl-glycine or 20 mM Tris HCl pH 7.0, 2 mM CaCl ² , for an appropriate time that can be determined experimentally and MS spectra visualizing deglycosylation will be obtained by analysis of rFVII a-xylosidase ESI-LC-MS (Q -STAR).

[0120] The resulting glycan remodeled rFVIIa will be purified from the enzyme a-xylosidase for example by anion exchange chromatography or gel filtration or suitable combinations of these. The purity of the prepared rFVIIa O-glycoform is verified by the rFVIIa O-glycopeptide map.

Example 5

Preparation of ^a truncated -xilosidasa for cloning and expression in E. coli

[0121] The crystal structure of YicI was recently resolved. Thus, cloning of a truncated α-xylosidase enzyme representing an active, catalytic domain of the YicI protein (or other similar α-xylosidases) may be possible and is in the pipeline, as a smaller enzyme, if activated, may better access the Xyl-Xyl-Glc-O-Ser52 present in native rFVIIa. A domain comprising the active site in the enzyme is provided from the structure. The gene sequence encoding this part of the YicI sequence is prepared from the existing YicI plasmid pET11a for an example by PCR amplification of relevant areas of the YicI gene, the primers used for PCR will have extensions with restriction enzyme sites that can be used for ligation of the truncated YicI gene in vector pET11a. The truncated enzyme, after expression and purification, will be evaluated for its potential for rFVIIa deglycosylation as described above.

Example 6

Preparation of rFVIIa with exclusively xylose-glucose at serine 52 or exclusively xylose-xylose-glucose at serine 52 by treatment ^to -xilosiltransferasa

Preparation of ^a -xilosiltransferasa

[0122] The enzyme, UDP-D-xylose: p-D-glucoside α-1,3-D-xylosyltransferase, can be obtained from HepG2 cells as described by Omichi et al. (1997). In short, HepG2 cells are grown in a medium supplemented with 10% fetal calf serum. The microsomal fraction is prepared by homogenizing the cells followed by centrifugation. The α-xylosyltransferase enzyme is purified by chromatography, for example on an anion exchange column and / or on other chromatographic columns known to the person skilled in the art. Fractions are collected during elution and fractions containing the enzyme α-xylosyltransferase are identified using the α-xylosyltransferase assay.

[0123] The enzyme, UDP-D-xylose: α-D-xyloside α1,3-xylosyltransferase, can be prepared from HepG2 cells as described by Minamida et al. (nineteen ninety six). In short, HepG2 cells are grown in a medium supplemented with 10% fetal calf serum. The microsomal fraction is prepared by homogenizing the cells followed by centrifugation. The α-xylosyltransferase enzyme is purified by chromatography, for example on an anion exchange column and / or on other chromatographic columns known to the person skilled in the art. Fractions are collected during elution and fractions containing the enzyme α-xylosyltransferase are identified using the α-xylosyltransferase assay.

Test ^to -xilosiltransferasa

[0124] Assays for α-xylosyltransferase are performed in an appropriate buffer, eg. ex. 10mM Hepes, pH 7.2, 0.1% Triton X-100, 0.5mM UDP-xylose (Sigma U5875), containing a suitable substrate, e.g. ex. the O-glycopeptides that can be obtained from the rFVIIa O-glycopeptide map or the pyridylaminated oligosaccharides prepared as described in Minamida et al. (Minamida et.al., Detection of UDP-D-xylose: a-D-xyloside a1-3xylosyltransferase activity in human hepatoma cell line HepG2. J. Biochem. 120 1002-1006, 1996). The reaction stops after an appropriate time, which can be determined experimentally, e.g. ex. by the addition of trifluoroacetic acid, and the test mixtures are analyzed by HPLC.

O-glycoforms of rFVIIa exclusively with xylose-glucose- in serine 52

[0125] The O-glycoforms of rFVIIa exclusively with xylose-glucose in serine 52 are obtained from (1) treatment of rFVIIa with xylosidase as described above, (2) purification of xylosidase-treated rFVIIa from xylosidase for example by anion exchange chromatography, and (3) by incubating xylosidase-treated rFVIIa in an appropriate buffer, e.g. ex. glycyl glycine, pH 7.0, 10 mM calcium chloride, with purified UDP-D-xylose: PD-glucoside α-1,3-D-xylosyltransferase and UDP-D-xylose for an appropriate time, which can be determined experimentally. The resulting glyco-remodeled rFVIIa is purified from UDP-D-xylose: enzyme α-D-glucoside α-1,3-D-xylosyltransferase for example by anion exchange chromatography. The purity of the prepared rFVIIa O-glycoform is verified by the rFVIIa O-glycopeptide map.

O-glycoforms of rFVIIa exclusively with xylose-xylose-glucose- in serine 52

[0126] O-glycoforms of rFVIIa with xylose-xylose-glucose in serine 52 are obtained from (1) treatment of rFVIIa with xylosidase as described above, (2) purification of xylosidase-treated xylosidase rFVIIa by example by anion exchange chromatography, (3) other treatment with UDP-D-xylose: aD-glucoside α-1,3-D-xylosyltransferase and UDP-D-xylose as described above, and (4) by incubation of the product in an appropriate buffer, e.g. ex. glycyl-glycine, pH 7.0, 10 mM calcium chloride, with purified UDP-D-xylose: a-D-xyloside, 1,3-xylosyltransferase, and UDP-D-xylose for an appropriate time, which can be determined experimentally. The resulting glyco-remodeled rFVIIa is purified from the UDP-D-xylose: enzyme α-D-xyloside α1,3-xylosyltransferase for example by anion exchange chromatography. The purity of the prepared rFVIIa O-glycoform is verified by the rFVIIa O-glycopeptide map.

Example 7

Analysis of the O-glycoform model of rFVIIa

Mapping of tryptic peptides of the rFVIIa light chain

[0127] The relative content of the O-glycoforms of rFVIIa is determined by mapping tryptic peptides of the rFVIIa light chain. The rFVIIa is reduced and alkylated and the rFVIIa light chain is purified on a RP-HPLC column eluted with a gradient of acetonitrile in water: trifluoroacetic acid. The light chain of purified rFVIIa is changed from buffer to Tris buffer, pH 7.5 and digested with trypsin. The tryptic digest of the rFVIIa light chain is analyzed on an RP-HPLC column (eg Nucleosil C18, 5 p, 300 A, 4.0 x 250 mM, Macherey-Nagel 720065) eluted with an acetonitrile gradient (0% -45% acetonitrile in 100 min) in water: trifluoroacetic acid (see figure 2). The flow is 1.0 ml / min and the detection is UV at 215 nm.

[0128] The maximum values containing the rFVIIa O-glycopeptides are eluted after approx. 60-65 min where the first and third maximum values contain O-glycopeptides with a serine-bound xylose-xylose-glucose 52, and the second and fourth maximum values contain O-glycopeptides with a serine-linked glucose 52.

[0129] Similarly, the first and second maximum values contain O-glycopeptides with a serine-bound tetrasaccharide 60, and the third and fourth maximum values contain O-glycopeptides with a serine-bound fucose 60.

Mapping of tryptic peptides of rFVIIa

[0130] The O-glycoform model can be analyzed by mapping tryptic peptides of rFVIIa. The rFVIIa is changed from buffer to Tris buffer, pH 7.5, and digested with trypsin. The tryptic digest of rFVIIa is analyzed on an RP-HPLC column (eg Nucleosil C18, 5 p, 300 A, 4.0 x 250 mM, Macherey-Nagel 720065) is eluted with a gradient of acetonitrile (0% -45% acetonitrile in 100 min) in water: trifluoroacetic acid. The flow is 1.0 ml / min and the detection is UV at 215 nm. The maximum values containing the rFVIIa O-glycopeptides are eluted after approx. 67-70 min where the second maximum value contains O-glycopeptides with a serine-linked xylosaxylose-glucose 52, and the first maximum value contains O-glycopeptides with a serine-linked glucose 52.

Analysis of the total mass of rFVIIa

[0131] The model of O-glycoforms can be analyzed by analysis of the total mass of rFVIIa. The rFVIIa is desalted on a Millipore ZipTip C4 column equilibrated with 1% formic acid and eluted with 3% formic acid in 90% methanol. The eluted sample is analyzed by the nanospray technique on a Qstar XL mass spectrometer.

The most important maximum value at approximately 50500 Da represents O-glycoforms of rFVIIa with a serine-linked glucose 52 and the most important maximum value at approximately 50800 Da represents O-glycoforms of rFVIIa with a serine-linked xylose-xylose-glucose 52.

Example 8

Purification of Glc-O-Ser52-FVII and Xyl-Xyl-Glc-O-Ser52-FVII

[0132] Glc-O-Ser52-FVII and Xyl-Xyl-Glc-O-Ser52-FVII was purified using two cycles of hydrophobic interaction chromatography (HIC). The column (1.0 cm in internal diameter x 7.0 cm length = 5.5 ml column volume (CV)) packed with Toso Haas TSK-Gel phenyl 5 PW, was equilibrated with 5 CV 10 mM histidine, 10 mM CaCl ² , 2.0 M NH4-acetate, pH 6.0. The column was loaded with approximately 2.5 mg of FVII pr. ml resin. To the loading solution, 2.0 M NH4-acetate and 10 mM CaCl ² were added before loading. The column was washed with 5 CV 10mM histidine, 10mM CaCl ² , 2.0M NH4-acetate, pH 6.0. Elution was performed using a 20 CV linear gradient of 10mM histidine, 10mM CaCl2, 2.0m NH4-acetate, pH 6.0 to 10mM histidine, 10mM CaCl ² , pH 6.0. The purification was carried out at a flow rate of 6 CV / h and at a temperature of 5 ° C. Fractions were collected during the elution.

[0133] FVII eluted at two major maximum overlap values (see Figure 4: HIC first cycle chromatogram). Fractions containing the first maximum value were pooled (fraction "A", figure 4) and purified again by a second HIC cycle, using the same chromatographic procedure as for the first HIC cycle (see figure 5: chromatogram obtained from reloading fraction "A" on the HIC column). The fractions containing the second most important maximum value (fraction "B", figure 4) were also pooled and purified again by a second HIC cycle, using the same chromatographic procedure as for the first HIC cycle (see figure 6: chromatogram obtained reloading the fraction "B" on the HIC column).

[0134] Glc-O-Ser52-FVII purified was identified in the maximum value fraction, fraction 10 (figure 5), obtained by recharging fraction "A" on the second HIC phase. Purified Xyl-Xyl-Glc-O-Ser52-FVII was identified in the maximum value fraction, fraction 15 (Figure 6), obtained by recharging fraction "B" on the second HIC phase. Identification was obtained by mapping tryptic peptides of rFVIIa as described in Example 7 (Figures 7A and 7B) and by analysis of the total mass of rFVIIa as described in Example 7 (Figures 8A and 8B). Both analyzes have shown a high content of Glc-O-Ser52-rFVIIa and a low content of Xyl-Xyl-Glc-O-Ser52-rFVIIa in the maximum value fraction, fraction 10, and a low content of Glc-O- Ser52-rFVIIa and a high content of Xyl- Xyl-Glc-O-Ser52-rFVIIa in the maximum value fraction, fraction 15. A quantification of the content of the O-glycoforms in the two maximum value fractions could not be obtained due to at a relatively low rFVIIa content in the fractions (Figures 7A and 7B: mapping of tryptic peptides: other fragments of rFVIIa peptides co-eluted with or eluted near O-glycopeptides, and the content of O-glycopeptides in low amounts therefore could not be determined.) (Figures 8A and 8B: analysis of total mass: other O- and / or N-glycoforms of rFVIIa, for example N-glycoforms of rFVIIa lacking N-acetylneuraminic acid, appeared in the spectra of mass, and the content of O-glycoforms of rFVIIa in low amounts therefore could not be be determined).

[0135] The specific activities of the maximum value fractions obtained from 1HIC (Table 1) were determined by the first generation coagulation test. Glc-O-Ser52-rFVIIa O-glycoform was found to have low specific activity while O-glycoform of Xyl-Xyl-Glc-OrSer52-rFVIIa had high specific activity. Table 1. Specific activities determined using the first generation coagulation assay for the maximum value fractions obtained from 1HIC. The rFVIIa content was determined by HPLC.

Example 9

Purification by hydrophobic interaction chromatography

[0136] Highly purified Glc-O-Ser52-rFVIIa preparations and highly purified Xyl-Xyl-Glc-O-Ser52-rFVIIa preparations can be obtained by repeated purification on hydrophobic interaction chromatography as described above. Preparations of highly purified Glc-O-Ser52-rFVIIa and Xyl-Xyl-Glc-O-Ser52-rFVIIa with a higher rFVIIa content can be obtained by increasing the amount of raw material for the hydrophobic interaction chromatography performed as described above. The content of each rFVIIa O-glycoform in highly purified preparations with a higher rFVIIa content can be quantified by mapping rFVIIa light chain tryptic peptides as described in Example 7. The specific activities of the highly purified can be determined by the first generation coagulation assay as mentioned above.

Claims (6)

1. Pharmaceutical composition comprising: (a) a preparation of a human factor VII polypeptide containing a Cys-X1-Ser-X2-Pro-Cys motif and where said serine is located at position Ser52 and forms part of a Glc-O-Ser covalent bond, where at least 85% of said serine 52 residues in the preparation are glycosylated with Xyl-Xyl-Glc; and (b) a pharmaceutically acceptable carrier. 2. Pharmaceutical composition according to claim 1, wherein at least 90%, at least 95%, or at least 98% of said serine residues 52 in the preparation are glycosylated with Xyl-Xyl-Glc. 3. Method of producing a preparation as described in claims 1-2, wherein the serine linked glycan is Xyl-Xyl-Glc; the method comprising the phases of: (a) obtaining a preparation of a human factor VII polypeptide containing a Cys-X1-Ser-X2-Pro-Cys motif and where said serine is located at position 52 and forms part of a Glc-O-Ser covalent bond; p. eg, from a genetically engineered cell (cell culture) or by isolation of the glycoprotein from a natural source; (b) contacting the preparation obtained in step (a) with UDP-D-xylose: pD-glucoside α-1,3-D-xylosyltransferase and an activated donor of xylosyl under appropriate conditions to transfer a xylose residue from the fraction xylose donor to the acceptor fraction, thereby producing the glycopeptide with an altered glycosylation pattern. (c) contacting the preparation obtained in step (b) with a UDP-D-xylose: aD-xyloside a-1,3-D-xylosyltransferase and an activated donor of xylosyl under appropriate conditions to transfer a xylose residue from the xylosyl donor fraction to the acceptor fraction, thereby producing the glycopeptide with an altered glycosylation pattern. 4. Method according to claim 3, further including the isolation phase of the preparation obtained in phase (b) before subjecting the preparation to phase (c). 5. Method according to any of claims 3-4, further including the isolation phase of the glycoprotein prepared in phase (c) with a glycosylation of xylose-xylose-glucose-O-serine / threonine. 6. Method according to any of claims 3-5, further including the phase of analysis of the structure of the oligosaccharides linked to the polypeptides to determine a glycoform pattern, and, optionally, repetition of phase (b) and / or the step (c) until the desired glycoform model is achieved.